WO2012040579A2 - Molecular screening across defined stiffness matrices - Google Patents

Abstract

This application related to methods for cell-based screening featuring substrates of user-defined stiffness. The methods may be used to assess the magnitude and character of cellular drug responses across a physiological stiffness range.

Description

MOLECULAR SCREENING ACROSS DEFINED STIFFNESS MATRICES

RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 61/385,942, filed September 23, 2010, which is incorporated herein by reference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0002] The invention was funded in part by the U.S. National Institutes of Health Grant HL092961. The United States Government has certain rights to the invention.

FIELD OF THE DISCLOSURE

[0003] The present invention relates to systems, compositions and methods for screening of cells.

BACKGROUND OF THE DISCLOSURE

[0004] The physical environment of a living cell influences its ability to proliferate, metabolize, differentiate, and remodel. Living cells specify lineage and express different phenotypic and physical states with extreme responsiveness to stiffness of their underlying matrix. The stiffness of the extracellular matrix is a vital mechanical cue that regulates cellular fate and function. Despite this recognition, the absence of a platform to efficiently study cells in physiologically relevant stiffness contexts has limited the pace of progress in this field. In particular, the possibility that substrate stiffness fundamentally alters cellular responses to biochemical effectors has major implications for cell-based research, but remains largely unexplored. As such, there is a need to integrate control of substrate stiffness into a standard multiwell format, emphasizing low cost, high throughput, and compatibility with conventional biological assays and detection methods.

SUMMARY OF THE INVENTION

[0005] Prior to the invention, cells, e.g., adherent cells, have been routinely cultured on rigid substrates that are typically orders of magnitude stiffer than their native tissue environments. The present disclosure provides methods for cell-based screening (e.g., using multiwell plates) featuring substrates of user-defined stiffness. The methods are used to assess the magnitude and character of cellular drug responses across a physiological stiffness range.

[0006] According to some embodiments, there is provided devices and methods related to a stiffness-tunable polymer substrate (e.g., polyacrylamide)-based high-throughput screening system. In some embodiments, there is provided methods of screening for candidate compounds (e.g., small molecules, macromolecules, proteins, nucleic acids, pharmacological agents, pharmacological inhibitors, etc.) that modulate stiffness-dependent biology (e.g., cell proliferation, cell adhesion, cell spreading, apoptosis, signaling events, and/or detection of soluble and insoluble factors). By a "candidate compound" is meant a chemical, protein, nucleic acid, or other agent, be it naturally-occurring or artificially-derived. Candidate compounds may include, for example, peptides, polypeptides, synthetic organic molecules, naturally occurring organic molecules, nucleic acid molecules, peptide nucleic acid molecules, and components and derivatives thereof. The term "pharmacological agent" or "pharmaceutical composition" is meant any composition, which contains at least one therapeutically or biologically active agent and is suitable for administration to the patient. Any of these formulations can be prepared by well-known and accepted methods of the art. See, for example, Remington: The Science and Practice of Pharmacy, 20th edition, (ed. A. R. Gennaro), Mack Publishing Co., Easton, Pa., 2000.

[0007] In some embodiments, there is provided a method for screening for compounds that have cell proliferation modulating activity comprising: contacting a cell or cell population with a test agent, wherein the cell or cell population is adherent to a hydrogel having a shear modulus of about 10 to about 1,000,000 Pascals (e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa, from about 10 to about 300,000 Pa, from about 10 to about 400,000 Pa, from about 10 to about 500,000 Pa, from about 10 to about 600,000 Pa, from about 10 to about 700,000 Pa, from about 10 to about 800,000 Pa, from about 10 to about 900,000 Pa); and determining whether cell proliferation activity is affected by the presence of the test agents as compared to a control. The method therefore is useful to identify agents that affect (e.g., inhibit or promote) cell proliferation or other cell activity in a physiologic setting because the cells are testes on substrates with an elastic modulus that corresponds to an in vivo microenvironment. [0008] In some embodiments, there is provided a method for screening for screening for compounds that have cell proliferation modulating activity comprising: placing cells or cell populations in assigned locations in an array of compliant substrates having a shear modulus of between about 10 to about 1,000,000 Pascals (e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa, from about 10 to about 300,000 Pa, from about 10 to about 400,000 Pa, from about 10 to about 500,000 Pa, from about 10 to about 600,000 Pa, from about 10 to about 700,000 Pa, from about 10 to about 800,000 Pa, from about 10 to about 900,000 Pa); contacting the cells or cell populations with a test agent; and determining whether cell proliferation activity of any one of the cell or cell populations of the array is affected by the presence of the test agents as compared to a control.

[0009] In some embodiments, there is provided a method for screening for compounds that inhibits cell proliferation comprising: placing a cell or cell population in assigned locations in an array of compliant substrates having a shear modulus of about 10 to about 1 ,000,000 Pascals (e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa, from about 10 to about 300,000 Pa, from about 10 to about 400,000 Pa, from about 10 to about 500,000 Pa, from about 10 to about 600,000 Pa, from about 10 to about 700,000 Pa, from about 10 to about 800,000 Pa, from about 10 to about 900,000 Pa); contacting the cell or cell population with a test agent; and determining which cell or cell population populations are rendered sensitive to drug treatment, thereby identifying a drug that inhibits cell proliferation.

[0010] In some embodiments, there is provided a method for screening for compounds that have cell adherence modulating activity comprising: contacting a cell or cell population with a test agent, wherein the cell or cell population is adherent to a hydrogel having a shear modulus of about 10 to about 1,000,000 Pascals (e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa, from about 10 to about 300,000 Pa, from about 10 to about 400,000 Pa, from about 10 to about 500,000 Pa, from about 10 to about 600,000 Pa, from about 10 to about 700,000 Pa, from about 10 to about 800,000 Pa, from about 10 to about 900,000 Pa); and determining whether cell adherence activity is affected by the presence of the test agents as compared to a control. The methods are used to identify compounds that modulate adherence in a physiological setting based on their affect on cells cultured on a substrate characterized by an elastic modulus mimicking the in vivo microenvironment.

[0011] In some embodiments, there is provided a method for screening for screening for compounds that have cell adherence modulating activity comprising: placing cells or cell populations in assigned locations in an array of compliant substrates having a shear modulus of between about 10 to about 1,000,000 Pascals (e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa, from about 10 to about 300,000 Pa, from about 10 to about 400,000 Pa, from about 10 to about 500,000 Pa, from about 10 to about 600,000 Pa, from about 10 to about 700,000 Pa, from about 10 to about 800,000 Pa, from about 10 to about 900,000 Pa); contacting the cells or cell populations with a test agent; and determining whether cell adherence activity of any one of the cell or cell populations of the array is affected by the presence of the test agents as compared to a control.

[0012] In some embodiments, there is provided a method for screening for compounds that inhibits cell adherence comprising: placing a cell or cell population in assigned locations in an array of compliant substrates having a shear modulus of about 10 to about 1 ,000,000 Pascals (e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa, from about 10 to about 300,000 Pa, from about 10 to about 400,000 Pa, from about 10 to about 500,000 Pa, from about 10 to about 600,000 Pa, from about 10 to about 700,000 Pa, from about 10 to about 800,000 Pa, from about 10 to about 900,000 Pa); contacting the cell or cell population with a test agent; and determining which cell or cell population populations are rendered sensitive to drug treatment, thereby identifying a drug that inhibits cell adherence.

[0013] In some embodiments, there is provided a method for screening for compounds that have cell spreading modulating activity comprising: contacting a cell or cell population with a test agent, wherein the cell or cell population is adherent to a hydrogel having a shear modulus of about 10 to about 1,000,000 Pascals (e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa, from about 10 to about 300,000 Pa, from about 10 to about 400,000 Pa, from about 10 to about 500,000 Pa, from about 10 to about 600,000 Pa, from about 10 to about 700,000 Pa, from about 10 to about 800,000 Pa, from about 10 to about 900,000 Pa); and determining whether cell spreading activity is affected by the presence of the test agents as compared to a control. [0014] In some embodiments, there is provided a method for screening for screening for compounds that have cell spreading modulating activity comprising: placing cells or cell populations in assigned locations in an array of compliant substrates having a shear modulus of between about 10 to about 1,000,000 Pascals (e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa, from about 10 to about 300,000 Pa, from about 10 to about 400,000 Pa, from about 10 to about 500,000 Pa, from about 10 to about 600,000 Pa, from about 10 to about 700,000 Pa, from about 10 to about 800,000 Pa, from about 10 to about 900,000 Pa); contacting the cells or cell populations with a test agent; and determining whether cell spreading activity of any one of the cell or cell populations of the array is affected by the presence of the test agents as compared to a control.

[0015] In some embodiments, there is provided a method for screening for compounds that inhibits cell spreading comprising: placing a cell or cell population in assigned locations in an array of compliant substrates having a shear modulus of about 10 to about 1 ,000,000 Pascals (e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa, from about 10 to about 300,000 Pa, from about 10 to about 400,000 Pa, from about 10 to about 500,000 Pa, from about 10 to about 600,000 Pa, from about 10 to about 700,000 Pa, from about 10 to about 800,000 Pa, from about 10 to about 900,000 Pa); contacting the cell or cell population with a test agent; and determining which cell or cell population populations are rendered sensitive to drug treatment, thereby identifying a drug that inhibits cell spreading.

[0016] In some embodiments, there is provided a method for screening for compounds that have apoptotic modulating activity comprising: contacting a cell or cell population with a test agent, wherein the cell or cell population is adherent to a hydrogel having a shear modulus of about 10 to about 1,000,000 Pascals (e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa, from about 10 to about 300,000 Pa, from about 10 to about 400,000 Pa, from about 10 to about 500,000 Pa, from about 10 to about 600,000 Pa, from about 10 to about 700,000 Pa, from about 10 to about 800,000 Pa, from about 10 to about 900,000 Pa); and determining whether cell viability is affected by the presence of the test agents as compared to a control. In some embodiments, the test agent is known to induce apoptosis in cells. [0017] In some embodiments, there is provided a method for screening for screening for compounds that have apoptotic modulating activity comprising: placing cells or cell populations in assigned locations in an array of compliant substrates having a shear modulus of between about 10 to about 1,000,000 Pascals (e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa, from about 10 to about 300,000 Pa, from about 10 to about 400,000 Pa, from about 10 to about 500,000 Pa, from about 10 to about 600,000 Pa, from about 10 to about 700,000 Pa, from about 10 to about 800,000 Pa, from about 10 to about 900,000 Pa); contacting the cells or cell populations with a test agent; and determining whether cell viability of any one of the cell or cell populations of the array is affected by the presence of the test agents as compared to a control. In some embodiments, the test agent is known to induce apoptosis in cells.

[0018] In some embodiments, there is provided a method for screening for compounds that induces apoptosis comprising: placing a cell or cell population in assigned locations in an array of compliant substrates having a shear modulus of about 10 to about 1,000,000 Pascals (e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa, from about 10 to about 300,000 Pa, from about 10 to about 400,000 Pa, from about 10 to about 500,000 Pa, from about 10 to about 600,000 Pa, from about 10 to about 700,000 Pa, from about 10 to about 800,000 Pa, from about 10 to about 900,000 Pa); contacting the cell or cell population with a test agent; and determining which cell or cell population populations are rendered sensitive to drug treatment, thereby identifying a drug that induces apoptosis. In some embodiments, the test agent is known to induce apoptosis in cells.

[0019] Also provided herein are methods of performing automated fluorescent imaging to detect, measure, and process data regarding morphology, proliferation, protein expression, and other parameters. These methods are carried out by placing a cell or cell population in assigned locations in an array of compliant substrates having a shear modulus of about 10 to about 1,000,000 Pascals (e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa, from about 10 to about 300,000 Pa, from about 10 to about 400,000 Pa, from about 10 to about 500,000 Pa, from about 10 to about 600,000 Pa, from about 10 to about 700,000 Pa, from about 10 to about 800,000 Pa, or from about 10 to about 900,000 Pa). For example, the substrates have a shear modulus of about 0.3 to about 55 kilo Pascals. Preferably, each location in the array of compliant substrates comprises a unique shear modulus. For example, the array of compliant substrates comprises a shear modulus that spans the entire stiffness range (e.g. , 0.3-55 kPa).

[0020] The cell or cell population stained or left unstained, and if stained, is fixed prior to staining the cell or cell population. Alternatively, the cell or cell population is not fixed prior to staining the cell or cell population. For example, the cells are stained for stained for F- actin and/or nuclei using methods well known in the art. Subsequently, the cell or cell population is imaged, thereby performing automated fluorescent imaging. Preferably, the cell or cell population is imaged using autofocusing to capture images of cells on all substrates. The images are detected and processed, e.g. , using a computer or other processor. For example, the cell or cell population is imaged using a Pathway HT™ fluorescence imaging system with autofocusing set at 400 nm step stage positioning. Imaging of the cell or cell population comprises imaging cellular morphology. The array comprises a 6 well plate, a 12 well plate, a 24 well plate, a 48 well plate, a 96 well plate, a 384 well plate, a 1536 well plate, a 3456 well plate, or a 9600 well plate.

[0021] Also provided are methods for screening for compounds that modulate cellular phenotype. These methods are carried out by placing cells or cell populations in assigned locations in an array of compliant substrates having a shear modulus of about 10 to about 1,000,000 Pascals (e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa, from about 10 to about 300,000 Pa, from about 10 to about 400,000 Pa, from about 10 to about 500,000 Pa, from about 10 to about 600,000 Pa, from about 10 to about 700,000 Pa, from about 10 to about 800,000 Pa, or from about 10 to about 900,000 Pa). The cells or cell populations are contacted with a nucleic acid composition, and it is determined whether cellular phenotype of any one of the cell or cell populations of the array is affected by the presence of the nucleic acid composition as compared to a control. For example, the nucleic acid composition is an inhibitor of ribonucleic acid (RNA) selected from the group consisting of small interfering RNA (siRNA) and short hairpin RNA (shRNA). Optionally, the phenotype comprises cellular morphology, cellular adhesion, or cytoskeleton function.

[0022] Methods of determining an individualized course of treatment of a subject are carried out by providing a cell population from a subject or providing a sample of cells obtained from an individual, e.g., a human patient, to be treated. Subsequently, the cells or cell population are placed in assigned locations in an array of compliant substrates having a shear modulus of between about 10 to about 1,000,000 Pascals (e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa, from about 10 to about 300,000 Pa, from about 10 to about 400,000 Pa, from about 10 to about 500,000 Pa, from about 10 to about 600,000 Pa, from about 10 to about 700,000 Pa, from about 10 to about 800,000 Pa, or from about 10 to about 900,000 Pa). The cells or cell population are contacted with a test agent, and cell viability, cell proliferation activity, adherence activity, or spreading activity of any one of the cells or cell populations of the array is detected, measured and analyzed, e.g., using a computer or other processor. Based on the collected and processed data, a determination is made as to whether any of the parameters are reduced or increased by the presence of the test agent compared to a control. The individualized course of treatment is selected base on performance of the test compound in the in vitro assay that simulates the in vivo condition. Such a determination is particularly useful for selecting treatment for abnormal conditions, e.g. , cancer, hypertension, fibrosis. In some cases, the test agent is administered to the subject to treat the abnormal condition, i.e. , disease.

[0023] Polynucleotides, polypeptides, or other agents are purified and/or isolated.

Specifically, as used herein, an "isolated" or "purified" nucleic acid molecule, polynucleotide, polypeptide, or protein, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. Purified compounds are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. For example, a purified compound is one that is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) of the desired compound by weight. Purity is measured by any appropriate standard method, for example, by column chromatography, thin layer chromatography, or high-performance liquid chromatography (HPLC) analysis. A purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) is free of the genes or sequences that flank it in its naturally-occurring state. Purified also defines a degree of sterility that is safe for administration to a human subject, e.g. , lacking infectious or toxic agents.

[0024] Similarly, by "substantially pure" is meant a nucleotide or polypeptide that has been separated from the components that naturally accompany it. Typically, the nucleotides and polypeptides are substantially pure when they are at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, free from the proteins and naturally-occurring organic molecules with they are naturally associated.

[0025] An "isolated nucleic acid" is a nucleic acid, the structure of which is not identical to that of any naturally occurring nucleic acid, or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes. The term covers, for example: (a) a DNA which is part of a naturally occurring genomic DNA molecule, but is not flanked by both of the nucleic acid sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner, such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybridgene, i.e., a gene encoding a fusion protein. Isolated nucleic acid molecules according to the present invention further include molecules produced

synthetically, as well as any nucleic acids that have been altered chemically and/or that have modified backbones.

[0026] A small molecule is a compound that is less than 2000 daltons in mass. The molecular mass of the small molecule is preferably less than 1000 daltons, more preferably less than 600 daltons, e.g., the compound is less than 500 daltons, 400 daltons, 300 daltons, 200 daltons, or 100 daltons.

[0027] The transitional term "comprising," which is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase

"consisting of excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of limits the scope of a claim to the specified materials or steps "and those that do not materially affect the basic and novel

characteristic(s)" of the claimed invention.

[0028] Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All published foreign patents and patent applications cited herein are incorporated herein by reference. Genbank and NCBI submissions indicated by accession number cited herein are incorporated herein by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] For a better understanding of the present invention, reference is made to the following description, taken in conjunction with the accompanying drawings, in which like reference characters refer to like parts throughout.

[0030] Figure 1. Integration of matrix stiffness into a multiwell plate for routine cell- based screening, (a) Hydrogel shear modulus (G) is specified by acrylamide:bisacrylamide content and measured by atomic force microscopy indentation (circles). The density of gel- bound collagen (lines) is tuned independently of stiffness, (b) Prototypical multiwell plate configuration, (c) Emergence of gradually increasing (blue), abrupt transition (green) and stiffness-insensitive (orange) cell accumulation following 72 hours of culture across a physiological stiffness range. A549, human lung adenocarcinoma cell line; NHDF, normal human dermal fibroblast; NHLF, normal human lung fibroblast; NIH 3T 3, mouse embryonic fibroblast cell line; MDCKII, Madin-Darby canine kidney epithelial cell line; hMSC, human mesenchymal stem cell; 16HBE14o-, human bronchial epithelial cell line; RLE6TN, rat lung epithelial cell line; L292, mouse fibroblast cell line; HEK29 3, human embryonic kidney cell line, (d) Effect of increasing collagen density on NHLF accumulation from 6- (blue) to 12- fold (red) over a minimal limit of detection (green), (e) Effect of increasing serum

concentration, (f) Increasing matrix stiffness promotes BrdU incorporation while suppressing caspase 3/7 activity in 10% serum. All error bars indicate mean + SD (n= 3).

[0031] Figure 2. Modification of drug responses by matrix stiffness, (a) Representative results from a small-scale drug screen. Error bars indicate mean + SD (n= 3). (b) Inhibiting non-muscle myosin II, ROCK or Rho promotes cell growth on 400 Pa substrates while suppressing growth on glass, (c) Effect of the indicated drugs over a range of matrix stiffness. (d) Effect of 1 pM GSK 429286 relative to no treatment on indexes of cell proliferation and apoptosis.

[0032] Figure 3. Cell type-specific responses to ROCK inhibition in differentially stiff contexts, (a) Demonstration of divergent responses in human adipose-derived mesenchymal stem cells (hASC), but only modestly differential responses in MDCKII cells, and stiffness- insensitive responses in HEK29 3 cells, (b) Effects on the accumulation of these cell types across a range of matrix stiffness, (c) Concurrent treatment of A549 cells with TGFI31 , or prior induction of epithelial-imesenchymal transition by 3 days treatment with TGFI31 , recapitulates divergent, stiffness-dependent growth responses to ROCK inhibition. Error bars indicate mean + SD (n= 3).

[0033] Figure 4. Schematic of polyacrylamide hydrogel incorporation into a multiwell plate.

[0034] Figure 5. Chemiluminesence-based detection of gel -bound collagen.

[0035] Figure 6 (a-c). Drug responses across two stiffness contexts. Coincident: the fold change in cell number occurring at the indicated dose of drug is not statistically different (p >0.05) between the 400 Pa and rigid context. Differential: drug responses between the two stiffness contexts are statistically different (p<0.05). Divergent: the effect of the indicated dose of drug on cell number is stimulatory in one context but inhibitory in the other, and both statistically different than 1 (p<0.05). Error bars are mean + s.d. (n=3). Specific p-values for each comparison are indicated and were evaluated by the two-tailed Student's t-test.

[0036] Figure 7. Divergent responses to ROCK inhibitors. The effects of Y-27632, fasudil hydrochloride and H-l 152 on the accumulation of NHLFs on 400 Pa versus rigid substrates. The divergent responses to GSK 429286 treatment are recapitulated in hMSCs and NIH3T3 cells.

[0037] Figure 8. ROCK inhibition rescues the cell spreading defect observed on low stiffness substrates. NHLFs were fixed following 48 hours of culture under the indicated conditions and stained for f-actin (green) and nuclei (blue).

[0038] Figure 9 is a bar chart showing the effect of siRNA targeting non-muscle myosin heavy chain Ila (MYH9) on fibroblasts.

[0039] Figure 10 is a series of photomicrographs illustrating automated imaging of cell morphology in a 384 well plate. Seven cell types cultured across increasing substrate stiffness, stained for F-actin (red) and nuclei (blue). Images were obtained at 200X magnification.

[0040] Figure 11 is a schematic illustrating an autofocusing imaging system to detect, measure, and process data regarding morphology, proliferation, protein expression, and other parameters. Cells are cultured in assigned locations in an array of compliant substrates. The cells are imaged utilizing an autofocusing microscope. Finally, a computer/processor is utilized to visualize, measure, and analyze physical parameters of cellular morphology.

DETAILED DESCRIPTION OF THE INVENTION

[0041] The physical environment of a living cell influences its ability to proliferate, metabolize, differentiate and remodel. Living cells specify lineage and express different phenotypic and physical states with extreme responsiveness to stiffness of their underlying matrix. The stiffness of the extracellular matrix is a vital mechanical cue that regulates cellular fate and function (Engler, A.J., Sen, S., Sweeney, H.L. & Discher, D.E. Cell 126, 677-689 (2006); Discher, D.E., Janmey, P. & Wang, Y. Science 310, 11 39-114 3 (2005)). Prior to the invention described herein, the absence of a platform to efficiently study cells in physiologically relevant stiffness contexts, which vary in tissue- and disease-specific manners, has limited the pace of progress in this field (Discher, D.E., Janmey, P. & Wang, Y. Science 310, 11 39-114 3 (2005); Levental, I., Georges, P.C. & Janmey, P.A. Soft Matter 3, 299- 306 (2007); Huang, S. & Ingber, D.E. Cancer Cell 8, 175-176 (2005)). In particular, the possibility that substrate stiffness fundamentally alters cellular responses to biochemical effectors has major implications for cell-based research, but was largely unexplored prior to the invention described herein (Rehfeldt, F., Engler, A.J., Eckhardt, A., Ahmed, F. &

Discher, D.E. Adv. Drug Deliv. Rev. 59, 1 329-1 339 (2007)).

[0042] In the following description of the preferred embodiment, reference is made to the accompanying drawings which form a part hereof, and in which is shown by way of illustration a specific embodiment in which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present invention.

[0043] In general, there is distinct stiffening (or softening) of tissue that occurs in a number of diseases, such as sclerodoma, atherosclerosis, emphysema, and fibrosis of the lung, liver and kidney. The compliant surface culture plate technology opens up the field to the assessment of stiffness-dependent cell behaviors at a level of detail that is not currently possible. For example, simulation of cell growth/behavior to contact with fat tissue is carried out by growing cells on a surface characterized by a shear modulus of fat (approximately 10 Pascal). Different tissue types are characterized by different stiffness, e.g., normal brain tissue has a shear modulus of approximately 200 Pascal. Cell growth/behavior also differs relative to the disease state of a given tissue, e.g., the shear modulus of normal mammary tissue is approximately 100 Pascal, whereas that of breast tumor tissue is approximately 2000 Pascal. Similarly, normal liver tissue has a shear modulus of approximately 300 Pascal compared to fibrotic liver tissue, which is characterized by a shear modulus of approximately 800 Pascal. Growth, signal transduction, gene or protein expression/secretion, as well as other physiologic parameters are altered in response to contact with different substrate stiffness and evaluated in response to contact with substrates characterized by mechanical properties that simulate different tissue types or disease states. Thus, the terms "soft substrate" and "stiff substrate" are relative terms depending upon the tissue type being tested. In screening for anti-tumor agents, candidate compounds are tested on cells growing on a substrate with an elastic modulus of a normal, non-cancerous tissue as well as on a substrate with an elastic modulus of a cancerous tissue of the same tissue or cell type (as well as points in between). Such tissue specific parameters are known in the art. For example, exemplary wells of a multiwall plate comprise wells with substrates that are 10, 20, 50, 100, 200, 400, 800, 1000, 2000, 5000, 10000, or more Pa as well as intermediate values.

[0044] The present invention provides for methods of screening for compounds (e.g. , small molecules, macromolecules, etc.) that modulate stiffness-dependent biology (e.g., cell proliferation, cell adhesion, cell spreading, apoptosis, signaling events, and/or detection of soluble and insoluble factors).

[0045] According to some embodiments, there is provided devices and methods related to a stiffness-tunable polyacrylamide high-throughput screening system. In some embodiments, there is provided methods of screening for compounds (e.g., small molecules,

macromolecules, etc.) that modulate stiffness-dependent biology (e.g., cell proliferation, cell adhesion, cell spreading, apoptosis, signaling events, and/or detection of soluble and insoluble factors).

[0046] According to some embodiments, methods are provided for screening for compounds that modulate stiffness-dependent biology comprising contacting a cell adherent to a compliant substrate having a shear modulus (i.e. , stiffness) of about 10 to about

3,000,000 Pascals (Pa) with a test agent and assessing the test agents ability to modulate a stiffness-dependent biology.

[0047] In some embodiments, cells are transferred into a suitable tissue culture plate (e.g., multiwell plate) having a surface covered with a compliant substrate having a shear modulus of about 10 to about 1,000,000 (e.g., from about 10 to about 100,000, from about 10 to about 150,000 Pa, from about 10 to about 200,000, from about 10 to about 300,000, from about 10 to about 400,000, from about 10 to about 500,000, from about 10 to about 600,000, from about 10 to about 700,000, from about 10 to about 800,000, from about 10 to about 900,000). Such plates are described in U.S. Application No. 12/675,839, incorporated herein by reference in its entirety. The cells are allowed to adhere to the compliant substrate. The cells are brought into contact with a test agent and after some incubation period, the effect of the test agent on a stiffness-dependent biology is assessed. For example, where cell proliferation is the stiffness-dependent biology, cells may be brought into contact with a test agent and after some incubation period (e.g., 72 hours) the impact of the test agent on the degree of proliferation of a cell or cell population is assessed.

[0048] In some embodiments, the cells are allowed to adhere to the compliant substrate in the presence or absence of test agent. For example, where cell adherence is the stiffness- dependent biology, cells may be brought into contact with a test agent at the time of plating, and after some incubation period (e.g., 72 hours) the impact of the test agent on the degree of adherence of a cell or cell population is assessed.

[0049] The incubation period may be from 4 hours to 96 hours (e.g., about 4, 8, 12, 24, 36, 48, 72, 96 hours and points in-between). In some embodiments, the cells are cultures for several days (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. days).

[0050] The effect of the test agent on a stiffness-dependent biology may be assessed in terms of a degree of impact such as a 10% to 1000% increase or decrease (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more increase or decrease in a specified activity).

[0051] According to some embodiments, there is provided a method for screening for compounds that have cell proliferation modulating activity comprising contacting a cell or population of cells with a test agent and detecting whether cell proliferation activity is affected. The cell or cell population are adherent cultures on the user-defined matrices (e.g., compliant substrate such as a hydrogel) of the present embodiments. According to some embodiments, the detecting step comprises detecting whether cell proliferation pathways have been modulated (e.g. , activated or inhibited).

[0052] According to some embodiments, there is provided a method for screening for compounds that have cell adhesion modulating activity comprising contacting a cell or population of cells with a test agent and detecting whether cell adhesion activity is affected. The cell or cell population are adherent cultures on the user-defined matrices (e.g., compliant substrate such as a hydrogel) of the present embodiments. According to some embodiments, the detecting step comprises detecting whether cell adhesion pathways have been modulated (e.g. , activated or inhibited).

[0053] According to some embodiments, there is provided a method for screening for compounds that have cell spreading modulating activity comprising contacting a cell or population of cells with a test agent and detecting whether cell spreading activity is affected. The cell or cell population are adherent cultures on the user-defined matrices (e.g., compliant substrate such as a hydrogel) of the present embodiments. According to some embodiments, the detecting step comprises detecting whether cell spreading pathways have been modulated (e.g. , activated or inhibited).

[0054] According to some embodiments, there is provided a method for screening for compounds that have apoptotic modulating activity comprising contacting a cell or population of cells with a test agent and detecting whether apoptotic activity is affected. The cell or cell population are adherent cultures on the user-defined matrices (e.g., compliant substrate such as a hydrogel) of the present embodiments. According to some embodiments, the detecting step comprises detecting whether apoptotic pathways have been modulated (e.g. , activated or inhibited).

[0055] According to some embodiments, cells (e.g., tumor cells) are plated into an assay dish. The assay dish may be a 6-well, 12-well, 24-well, 96-well, 384-well or 1536-well assay dish. The cells are contacted with a selected drug to be screened and the cell response to selected drug is assessed (e.g., viability or sensitivity).

[0056] The arrays can be designed to take advantage of systems developed for current assay formats, such as detection systems and robotic systems and the like which are designed to handle 6-well, 12-well, 24-well, 96-well, 384-well, 1536-well plates, or even 9,600- microwell plates, for example. The present invention is not limited to the presently used microtiter plate configurations but provides for any configuration necessary to take advantage of industry standards as well as provides the flexibility to design for novel configurations. Tissue Culture Substrate/Solid Support - Mutiwell Systems

[0057] In general, in another aspect, the invention provides an apparatus including a tissue culture substrate or solid substrate suitable for tissue culture (e.g., multi- well plate) and a gel affixed to a surface of the plate. The tissue culture substrate may be any surface or solid support suitable for use in tissue culture. In some embodiments, the tissue culture substrate is in the form of multiwell, glass-bottom plates. Methods for assembly fabricating hydrogels on the surface of a tissue culture substrate/solid support (e.g., culture dishes with glass-bottom plates) are provided for in WO 2009/032164, incorporated herein by reference in its entirety.

[0058] In some embodiments, the thickness of the gel is less than 1 millimeter (e.g., less than 800 μιη, less than 600 μιη, less than 500 μιη, less than 400 μιη, less than 300 μιη, less than 200 μιη, less than 150 μιη, less than 100 μιη, less than 75 μιη, less than 50 μιη, etc.). In some embodiments, the thickness of the gel is between 50μιη and 150 μιη (e.g., ΙΟΟμιη or less than 100 μιη).

[0059] In some embodiments, multiple shear modulus gels, varying in orientation, are casted and derivatized in a multi-well glass-bottom plate. In some embodiments, a collection of wells with varying shear modulus (i.e., stiffness) is provided. For example, a multi-well plate is fabricated with a range of compliant substrates and used to test cellular responses across a plate with well each of which contain a substrate with stiffness ranging from e.g., 50 to 150,000 Pascals. For example, lung fibroblast proliferation and apoptosis which is strongly dependent upon substrate shear modulus can be tested. Cells can be grown in the plates, and can be manipulated and analyzed in a manner consistent with conventional multi-well plates. Modern (circa 2008) microplates generally have either 384, 1536, or 3456 wells. These are all multiples of 96, reflecting the original 96 well microplate with 8 x 12 9mm spaced wells.

[0060] Multiple shear modulus gels, varying in orientation, can be casted and derivatized in a multi-well glass-bottom plate. In some embodiments, a collection of wells with varying shear modulus (i.e., stiffness) can be provided.

[0061] In some embodiments, there is provided an apparatus including a tissue culture substrate or solid substrate suitable for tissue culture (e.g., multi- well plate) and a gel affixed in at least a first well in the plate. [0062] In some embodiments, a multi- well plate can use synthetic matrix-coated hydrogels to span a physiological range of shear modulus values. For example, a 96-well plate, which is a generally used format for biological assays, can be used. The system can also be extended to other formats that are amendable to high- throughput screening (384- well plates or other multiple of 96) or cell cultivation (petri dish). In general, to replicate soft tissue elasticity, a polyacrylamide hydrogel is polymerized as a thin, optically transparent layer which is affixed to the bottom of each well. By controlling the number of crosslinks that interconnect the hydrogel network, the elasticity can be tuned over the range of typical soft tissues (heart, lung, kidney, liver, muscle, neural, etc.) from elastic modulus -20 Pascals (fat) to -100,000 Pascals (skeletal muscle) or more. In comparison, prior art cell culture dishes made from polystrene plastic have a stiffness of -3,000,000,000 Pascals.

[0063] The well can be a multi-well configuration such as a 96- well assembly comprising a 12 x 8 matrix of wells in a plate (e.g., a Matrical 96-well assembly). The well can also be a 6-well, 24- well, 384-well configurations. Generally, the well can include standard multi-well plates used to study various biological endpoints under different interventions. The bottom of the well bottom can be glass to allow for observation of cells placed within the well.

User Defined Matrices

[0064] The present invention provides for methods for fabricating user defined matrices (e.g., hydrogels) with elastic properties covering a broad, physiologically relevant range. The hydrogel solution can be delivered unto a surface of the tissue culture substrate (e.g., well) via a dispensing system (e.g., pipette, automated liquid dispensing system). Different concentrations of the hydrogel solution can be used to produce gels of different shear modulus, for example, ranging from 20 to 100,000 Pa.

[0065] Polymer hydrogels suitable for use with the present embodiments include, but are not limited to, the following: acrylamide/bis-acrylamide, polyalkylimide, poly(N- vinyl formamide), polyvinyl alcohol, poly(ethylene glycol), polydimethylsiloxane, silicone, glycosaminoglycans, hyaluronic acid, chondroitin sulfate, polysaccharide, self-assembling peptides, collagen, gelatin, fibrin, methylcellulose, and agarose.

[0066] In some embodiments, the polymerization solution can be comprised of variable ratios of acrylamide:bis-acrylamide, and can be delivered into the well via a dispensing system (e.g., pipette, automated liquid dispensing system). Different concentrations of the acrylamide / bis-acrylamide mixture can be used to produce gels of different shear modulus, for example, ranging from 20 to 100,000 Pa.

[0067] In an embodiment, hydrogels can be affixed (i.e., firmly attached) to the bottom of a well (e.g., wells in 24, 96, 384 well plates). The firm attachment enables long-term cell cultures, as well as compatibility with some assay reagents that may cause the hydrogels to shrink and detach. The plates can support the attachment and growth of different cell types and can be compatible with standard multi-well plate assays. The mechanical properties of the hydrogels can be reproducible and stable to increase the shelf life of the substrate.

[0068] In some embodiments, the hydrogel solution is delivered unto a surface of a well such that wells can be covered with gels of uniform thickness and static or various shear moduli. A 96- well plate, for example, can be loaded with plurality of polymerization solutions in different wells. The wells can be covered with gels of uniform thickness and various shear moduli (e.g., 100, 200, 400, 800, 1600, 3200, 6400, 12800, 25600, and 51200 Pa). For example, the well columns can be loaded with gels of increasing shear modulus in ascending order (i.e., 50, 100, 200, 400, 800, 1600, 3200, 6400, 12800, 25600 and 51200 Pa respectively).

[0069] In some embodiments, the hydrogels are optically transparent, thin (<100 μιη) and bound covalently to the entire surface of each well. To achieve a gel thickness of less than 100 μιη for a 384-well assembly, each well can typically receive 1-2 microliters of polymerization solution. A 96- well assembly typically will receive about 5 microliters. The amount of polymerization solution can change based on the desired thickness of resulting gel. The delivery system can be a pipette or similar liquid dispensing system (e.g., BioTek Microplate Liquid dispensing system). The glass plate can be hydrophobic glass. As an example, and not a limitation, a circular well, the diameter of the glass plate is less than the diameter of the well.

[0070] In some embodiments, methods of fabricating hydrogels includes placing a first polymerizing solution into a well, covering the first polymerizing solution with a plate, such that the area of the plate is less than the area of the well, conjugating the first polymerizing solution with a ligand, placing a second polymerizing solution into the well, such that oxygen in the air substantially inhibits polymerization at the air-liquid interface; and detoxifying the well. A second polymerizing solution can be distributed evenly by tapping the well. The second polymerizing solution can substantially cover a ring shaped area defined by the edge of the first polymerizing solution and the well. The second polymerizing solution can affix the first polymerizing solution to the well.

[0071] Acrylamide, and other polymer solutions, can be highly toxic to cells, so it is generally necessary to include a detoxification process. For example, glutathione can be added to detoxify free, unpolymerized acrylamide (e.g., a solution of glutathione can be dispensed into the well and incubated for several hours prior to seeding the well with cells). Shear Moduli

[0072] In some embodiments, the shear modulus of the gels can cover a range or be kept static. In some embodiments, a multi-well tray can include gels with a standard shear modulus (e.g., 5000, 10,000, 17,000 Pa) in each well.

[0073] In some embodiments, the configuration of wells encompassing the stiffness range of adipose tissue to skeletal muscle can be specified. Preferably, the shear modulus of the cell culture substrate of the device is in the range of the tissue type to be evaluated.

[0074] The shear modulus may range from about 10 to about 3,000,000,000 Pascals (Pa). In some embodiments, the shear modulus may range from about 10 to about 100,000 Pascals (Pa), including from aboutlO Pa to about 100,000 Pa, from about 20 Pa to about 100,000 Pa, from about 100 Pa to about 100,000 Pa, from about 400 Pa to about 100,000 Pa, from about 800 Pa to about 100,000 Pa, from about 1200 Pa to about 100,000 Pa, from about 1600 Pa to about 100,000 Pa, from about 3200 Pa to about 100,000 Pa, from about 6400 Pa to about 100,000 Pa, from about 12800 Pa to about 100,000 Pa, from about 25600 Pa to about 100,000 Pa, from about 50,000 Pa to about 100,000 Pa, from about 10 Pa to about 51200 Pa, from about 20 Pa to about 51200 Pa, from about 100 Pa to about 51200 Pa, from about 400 Pa to about 51200 Pa, from about 800 Pa to about 51200 Pa, from about 1200 Pa to about 51200 Pa, from about 1600 Pa to about 51200 Pa, from about 3200 Pa to about 51200 Pa, from about 6400 Pa to about 51200 Pa, from about 12800 Pa to about 51200 Pa, from about 25600 Pa to about 51200 Pa, from about 10 Pa to about 25600 Pa, from about 20 Pa to about 25600 Pa, from about 100 Pa to about 25600 Pa, from about 400 Pa to about 25600 Pa, from about 800 Pa to about 25600 Pa, from about 1200 Pa to about 25600 Pa, from about 1600 Pa to about 25600 Pa, from about 3200 Pa to about 25600 Pa, from about 6400 Pa to about 25600 Pa, from about 12800 Pa to about 25600 Pa, from about 10 Pa to about 15000 Pa, from about 20 Pa to about 15000 Pa, from about 100 Pa to about 15000 Pa, from about 400 Pa to about 15000 Pa, from about 800 Pa to about 15000 Pa, from about 1200 Pa to about 15000 Pa, from about 1600 Pa to about 15000 Pa, from about 3200 Pa to about 15000 Pa, from about 6400 Pa to about 15000 Pa, from about 12800 Pa to about 15000 Pa, from about 10 Pa to about 9000 Pa, from about 20 Pa to about 9000 Pa, from about 100 Pa to about 9000 Pa, from about 400 Pa to about 9000 Pa, from about 800 Pa to about 9000 Pa, from about 1200 Pa to about 9000 Pa, from about 1600 Pa to about 9000 Pa, from about 3200 Pa to about 9000 Pa, from about 6400 Pa to about 9000 Pa, from about 10 Pa to about 5000 Pa, from about 20 Pa to about 5000 Pa, from about 100 Pa to about 5000 Pa, from about 400 Pa to about 5000 Pa, from about 800 Pa to about 5000 Pa, from about 1200 Pa to about 5000 Pa, from about 1600 Pa to about 5000 Pa, from about 3200 Pa to about 5000 Pa, from about 10 Pa to about 2000 Pa, from about 20 Pa to about 2000 Pa, from about 100 Pa to about 2000 Pa, from about 400 Pa to about 2000 Pa, from about 800 Pa to about 2000 Pa, from about 1200 Pa to about 2000 Pa, from about 1600 Pa to about 2000 Pa, from about 10 Pa to about 1000 Pa, from about 20 Pa to about 1000 Pa, from about 100 Pa to about 1000 Pa, from about 400 Pa to about 1000 Pa, from about 800 Pa to about 1000 Pa, from about 10 Pa to about 500 Pa, from about 20 Pa to about 500 Pa, from about 100 Pa to about 500 Pa, from about 400 Pa to about 500 Pa, from about 10 Pa to about 400 Pa, from about 20 Pa to about 400 Pa, from about 100 Pa to about 400 Pa, from about 10 Pa to about 200 Pa, from about 20 Pa to about 200 Pa, from about 100 Pa to about 200 Pa, from about 300 Pa to about 500 Pa, and from about 300 Pa to about 2000 Pa. This includes a shear modulus of one or more of the following: 10, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 400, 800, 1600, 3200, 6400, 12800, 25600, and 51200 Pa).

Attachment Ligands

[0075] In some embodiments, the hydrogels may be further modified or treated with a substance (e.g., matrix protein) to foster cell attachment. For example, to enable cell attachment, the otherwise inert gels may be functionalized with equivalent amounts monomeric collagen or other suitable attachment ligand or combinations of attachment ligands. Suitable attachment ligands include, but are not limited to, the following: collagen I, collagen IV, fibronectin, vitronectin, laminin, or RGD peptides. Attachment ligands can be coupled to the gel surface using any means known in the art. The uniformity of ligand binding can be assessed with anti-ligand and anti-IgG-coated fluorescent beads.

[0076] The attachment ligand (e.g., collagen) can be fixed at a density between 1 μg/ml and 100 μg/ml (e.g., between 1 μg/ml and 100 μg/ml, between 1 μg/ml and 80 μg/ml, between 1 μg/ml and 60 μg/ml, between 1 μg/ml and 50 μg/ml, between 1 μg/ml and 40 μg/ml, between 1 μg/ml and 20 μg/ml, between 1 μg/ml and 10 μg/ml, between 1 μg/ml and 5 μg/ml, between 2 μg/ml and 100 μg/ml, between 2 μg/ml and 80 μg/ml, between 2 μg/ml and 60 μg/ml, between 2 μg/ml and 50 μg/ml, between 2 μg/ml and 40 μg/ml, between 2 μg/ml and 20 μg/ml, between 2 μg/ml and 10 μg/ml, between 2 μg/ml and 5 μg/ml, between 5 μg/ml and 100 μg/ml, between 5 μg/ml and 80 μg/ml, between 5 μg/ml and 60 μg/ml, between 5 μg/ml and 50 μg/ml, between 5 μg/ml and 40 μg/ml, between 5 μg/ml and 20 μg/ml, between 5 μg/ml and 10 μg/ml, and between 5 μg/ml and 15 μg/ml)

Serum

[0077] The concentration of serum present in the culture media may be optimized between 0% and 15% for each test condition (e.g., between 1% and 10%, between 3% and 10%, between 5% and 10%, between 1 % and 15%, between 3% and 15%, between 5% and 15%, between 10% and 15%, between 1 % and 3%, between 1 % and 5%, between 3% and 5%, etc.) Cells

[0078] Any cell type known in the art may be used and studied with the methods and systems of the present embodiments. The cells may be primary cells or of an established cell line. Cells may be of a type selected from the group consisting of cardiovascular, gastrointestinal, kidney, genitourinary, musculoskeletal, nervous system, oral, breast, periodontal, or skin cell or progenitor thereof. Cells may be normal or transformed cells.

[0079] In some embodiments, the cells are tumor cells of known genotype and/or representative of a particular form of cancer.

[0080] In some embodiments, cells are seeded on a hydrogel (e.g., rapidly casted hydrogels) of specified stiffness over a tissue culture substrate. In some embodiments, cells are seeded at subconfluent density (e.g., less than 100 cells/mm², less than 90 cells/mm², less than 80 cells/mm², less than 70 cells/mm², less than 60 cells/mm², less than 50 cells/mm², less than 40 cells/mm², less than 30 cells/mm², less than 20 cells/mm², less than 15 cells/mm², less than 10 cells/mm², less than 5 cells/mm², and points in between).

[0081] Cells grown in the multiple shear modulus plate can be fixed and immunologically stained, or isolated for gene expression and protein analysis. Attachment-dependent cell types can be studied, including fibroblasts, smooth muscle, endothelial, epithelial, tumor, osteoid, and neuronal. The plate can serve as a tool to direct the differentiation of adult or embryonic stem cells. [0082] In some embodiments, the cell is a stem cell or progenitor cell such as an embryonic stem cell or induced pluripotent stem cell. Such a stem cell may be a pluripotent cell of mesodermal, ectodermal or endodermal origin. In some embodiments, a stem cell is of mesodermal origin. In some embodiments, a stem cell is a hematopoiteic progenitor cell. Exemplary cells to be used in screening methods include endothelial cells, endothelial precursor cells, endothelial progenitor cells, macrophages, fibroblasts, pericytes, smooth muscle cells, ASCs, preadipocytes, differentiated or de-differentiated adipocytes, keratinocytes, unipotent and multipotent progenitor and precursor cells, as well as lymphocytes and precursors thereof. In some embodiments, the cell is a cancer (tumor) cell, e.g., lung, breast, colon, prostate, pancreas, stomach, liver, brain, kidney, uterus, cervix, ovaries, urinary tract, rectal tract, or is a melanoma or leukemia. In some embodiments, the cell is a non-tumor (normal) tissue derived cell or lineage committed cell, e.g. a breast, liver, prostate, brain, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, muscle, thyroid, prostate, uterus, cervix, ovaries, urinary tract, rectal tract, pancreatic or bladder cell. In some embodiments, the cell is a patient-derived primary cells (e.g., tumor-derived, non- tumor (normal) tissue derived).

Test agents

[0083] The selected drug or test agents may be any drug known to interact with cell proliferative pathways, cell adhesion pathways, cell spreading pathways, apoptotic pathways, etc.

[0084] In some embodiments, the selected drug or test agent is a small molecule. Small molecules include, but are not limited to, peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic and inorganic compounds (including heterorganic and organomettallic compounds) having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 2,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

[0085] In some embodiments, a small molecule is a compound that is less than 2000 daltons in mass (e.g., a small molecule inhibitor). The molecular mass of the small molecule compounds is preferably less than 1000 daltons, more preferably less than 600 daltons, e.g., the compound is less than 500 daltons, 400 daltons, 300 daltons, 200 daltons, or 100 daltons.

[0086] In addition to screening small molecule compounds (e.g., inhibitors) to identify drugs that are active in physiologically relevant settings (on cells residing in

microenvironments the correspond to the substrate properties of the screening assay), the methods are useful to identify gene or gene products/proteins that govern substrate or microenvironment stiffness dependency. For example, a RNA interference (RNAi) (e.g. , small interfering RNA) screen is used to knockdown genes and cell behavior on user-defined substrates evaluated to determine genes that play a role in stiffness dependency. Exemplary genes to be target include those that are involved in cell adhesion and cytoskeleton function. Alternatively, pharmacological agents are utilized to inhibit genes of interest.

[0087] In another example, antibodies, e.g., neutralizing antibodies that bind to cell surface proteins such as integrins or growth factor receptors, are screened to identify those cell surface proteins or structures that play a role in cell stiffness dependency. The expression of such genes/gene products are unique to substrate stiffness characteristics and can identify molecules that are differentially expressed on diseased cells compared to normal cells. For example, a cell surface protein is present on diseased cells but not normal cells or are expressed. Such proteins are then exploited for targeted drug delivery strategies.

[0088] In some embodiments, the selected drugs or test agent is a known

chemotherapeutic agent. The term "chemotherapeutic agent" is art recognized and is intended to include those chemical and biological agents, including small molecules and larger molecules, such as peptides, proteins, lymphokines, antibodies, tumor necrosis factor, conjugates of antibodies with toxins, and other chemical or biological molecules which have an anti-tumor effect (e.g., inhibits cancer cell growth such as by inducing cancer cell senescence or death).

[0089] Chemotherapeutic agents to be screened fall into the following exemplary categories: modulators of intracellular signaling proteins, alkylating agents, antimetabolites, antitumor antibiotics, plant alkaloids, terpenoids, topoisomerase inhibitors, taxanes, corticosteroid hormones, mitotic inhibitors, and nitrosoureas, hormone agents, miscellaneous agents, and any analog or derivative variant thereof. See also Table 1-1 in Example 1 below.

[0090] Common chemotherapeutic agents include, but are not limited to, Sorafenib (4- [4- [[4-chloro-3-(trifluoromethyl)phenyl]carbamoylamino] phenoxy]-N-methyl-pyridine-2- carboxamide), Gefitinib (N-(3-chloro-4-fluoro-phenyl)-7-methoxy-6-(3-morpholin-4- ylpropoxy)quinazolin-4-amine) , Imatinib (4- [(4-methylpiperazin- 1 -yl)methyl] -N- [4-methyl- 3-[(4-pyridin-3-ylpyrimidin-2-yl)amino]phenyl]benzamide), PLX5568, Dasatinib (BMS- 354825), Staurosporine, midostaurin, 5-fluorouracil, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin (CDDP), cyclophosphamide, dactinomycin,

daunorubicin, doxorubicin, estrogen receptor binding agents, etoposide (VP16), farnesyl- protein transferase inhibitors, gemcitabine, ifosfamide, mechlorethamine, melphalan, mitomycin, navelbine, nitrosurea, plicomycin, procarbazine, raloxifene, tamoxifen, taxol, temazolomide (an aqueous form of DTIC), transplatinum, vinblastine and methotrexate, vincristine, or any analog or derivative variant of the foregoing. (See US Publication No. 2008/0089860).

[0091] Imatinib is used to treat chronic myelogenous leukemia (CML), gastrointestinal stromal tumors (GISTs) and other cancers.

Modulators of intracellular signaling proteins

[0092] Chemotherapeutic agents include inhibitors or modulators of intracellular signaling proteins. Categories of such agents include: protein kinase inhibitors; Src family tyrosine kinases inhibitors; BCR/ABL inhibitors; MEK inhibitors; Raf inhibitors; SH2 55 inhibitors; PI3K inhibitors; and JNK inhibitors. The use of these inhibitors may be selected based on the target cell being screened. For example, MEKi in melanoma, PI3K inhibitors in lung cancer cell lines.

[0093] Compositions to be screened include small molecules (e.g., less than 2,000 daltons molecular mass), antibodies, or polynucleotides (e.g., RNAi molecules). The table below provides a list of protein kinase inhibitors.

[0094] Dasatinib is dual BCR/ABL and Src family of tyrosine kinases inhibitors may be used in chronic myelogenous leukemia (CML) and metastatic melanoma.

MEK inhibitors (MEKi)

[0095] MEK is a key protein kinase in the RAS/RAF/MEK/ERK pathway, which signals for cancer cell proliferation and survival. MEK is frequently activated in cancer, in particular in tumors that have mutations in the RAS and RAF oncogenes. MEK also regulates the biosynthesis of the inflammatory cytokines TNF, IL-6 and IL-1, which can act as growth and survival factors in cancer.

[0096] Examples of MEK inhibitors include: 2-(2-amino-3-methoxyphenyl)-4-oxo-4H- [l]benzopyran; 2-(2-chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro- benzami- de; ARRY-162; ARRY-300; AZD6244 (ARRY-886); and AZD8330 (ARRY-704). Categories of MEK inhibitors include: Substituted diarylamines (U.S. Patent No. 7001905); 4-arylamino, 4-aryloxy, and 4-arylthio diarylamines and derivatives (U.S. Patent No.

6506798); benzenesulfonamide derivatives (U.S. Patent No. 6440966); Benzoheterocycles (U.S. Patent No. 6469004); Substituted 3-cyanoquinolines (U.S. Patent No. 7173135); 4- Bromo or 4-iodo phenylamino benzhydroxamic acid derivatives (U.S. Patent No. 6821963); N3 alkylated benzimidazole derivatives (U.S. Patent No. 7235537); Amino-thio-acrylonitriles (U.S. Patent No. 6703420); Quinoline derivatives (U.S. Patent No. 6809106);

Sulohydroxamic acids and sulohyroxamates (U.S. Patent No. 6455582). The disclosures of the above referenced patents are incorporated herein by reference in their entireties.

Additional MEK inhibitors are disclosed in U.S. Publication No. 2003/0055095, which is incorporated by reference herein in its entirety. PI3K inhibitors (PI3Ki)

[0097] Examples of PI3K inhibitors are Wortmannin (Calbiochem, La Jolla, Calif.; Cat. No. 681675), benzo[b]thiophene-2-carboxamide, 5,6-dimethoxy-3-phenoxy-N-lH-tetrazol-5- yl-. Specifically, any compound is a PI3K inhibitor if it is able to block the catalytic activity of PI3K. Other PI3K inhibitors known in the art can also be utilized in the present invention. JNK inhibitors (JNKi)

[0098] Examples include: Anthra[l,9-cd]pyrazol-6(2H)-one (selective c-Jun N-terminal protein kinase (JNK) inhibitor).

Alkylating Agents

[0099] Alkylating agents are drugs that directly interact with genomic DNA to prevent the cancer cell from proliferating. This category of chemotherapeutic drugs represents agents that affect all phases of the cell cycle, that is, they are not phase-specific. An alkylating agent, may include, but is not limited to, a nitrogen mustard, an ethylenimene, a

methylmelamine, an alkyl sulfonate, a nitrosourea or triazines. They include but are not limited to: busulfan, chlorambucil, cisplatin, carboplatin, chlorambucil, cyclophosphamide (Cytoxan), dacarbazine, ifosfamide, mechlorethamine (mustargen), Melphalan (PAM), oxaliplatin, cis-Diammminedichloroplatinum (II) (CDDP), nitrosoureas such as N,N'-bis(II- chloroethyl)-N-nitrosourea (BCNU), nitrogen mustards, ethyleneimine compounds, alkyl sulphonates, cisplatin and dacarbazine.

Antimetabolites

[00100] Antimetabolites disrupt DNA and RNA synthesis. Unlike alkylating agents, antimetabolites specifically influence the cell cycle during S phase. Antimetabolites can be differentiated into various categories, such as folic acid analogs, pyrimidine analogs and purine analogs and related inhibitory compounds. Antimetabolites include but are not limited to, 5-fluorouracil (5-FU), cytarabine (Ara-C), fludarabine, gemcitabine, methotrexate, folic acid, purine or pyrimidine antagonists, 6-Mercaptopurine, fluorodeoxyuridine, cytosine arabinoside, azathioprine, mercaptopurine and thioquinone.

Natural Products

[00101] Natural products generally refer to compounds originally isolated from a natural source, and identified as having a pharmacological activity. Such compounds, analogs and derivatives thereof may be isolated from a natural source, chemically synthesized or recombinantly produced by any technique known to those of skill in the art. Natural products include such categories as mitotic inhibitors, antitumor antibiotics, enzymes and biological response modifiers.

[00102] Mitotic inhibitors include plant alkaloids and other natural agents that can inhibit either protein synthesis required for cell division or mitosis. They operate during a specific phase during the cell cycle. Mitotic inhibitors include, for example, docetaxel, etoposide (VP16), teniposide, paclitaxel, taxol, vinblastine, vincristine, and vinorelbine.

[00103] Taxoids are a class of related compounds isolated from the bark of the ash tree, Taxus brevifolia. Taxoids include, but are not limited to, compounds such as docetaxel and paclitaxel. Paclitaxel binds to tubulin (at a site distinct from that used by the vinca alkaloids) and promotes the assembly of microtubules.

[00104] Vinca alkaloids are a type of plant alkaloid identified to have pharmaceutical activity. The vinca alkaloids include: Vincristine, Vinblastine, Vinorelbine, and Vindesine.

[00105] Podophyllotoxin is a plant-derived compound which is said to help with digestion as well as used to produce two other cytostatic drugs, etoposide and teniposide.

Antitumor Antibiotics

[00106] Antitumor antibiotics have both antimicrobial and cytotoxic activity. These drugs also interfere with DNA by chemically inhibiting enzymes and mitosis or altering cellular membranes. These agents are not phase specific and therefore work in all phases of the cell cycle. Examples of antitumor antibiotics include, but are not limited to, actinomycin, adriamycin, bleomycin, dactinomycin, daunorubicin, doxorubicin (Adriamycin), plicamycin (mithramycin) and idarubicin.

Nucleic acids

[00107] Cancer often involves mutant genes that promote uncontrolled cell growth. In the last few years, researchers have silenced more than a dozen known cancer-causing genes with RNAi. Most of this success has been with cell cultures in the lab, and delivery poses the key hurdle in moving from the lab to the bedside of patients. RNAi drugs and other drugs that affect genes involved in tumorigenesis and metastasis may have different effects on cells depending on the matrix stiffness upon which they are tested (reflective of the in vivo environment). Thus, the compositions and methods described herein are used to evaluate how nucleic acids (e.g., RNAi and other nucleic acid-based therapies) and other drugs reach, penetrate, and affect tumors. [00108] RNAi is a naturally occurring mechanism that controls gene expression at the post-transcriptional level. In eukaryotes, double-stranded interfering RNAs target complementary mRNAs for degradation, resulting in selective silencing of specific proteins. This characteristic of RNAi makes it a valuable laboratory research tool, both in cells and in whole animal models. The development of RNAi libraries, which are composed of reagents that systematically target every gene in the genome, has made it possible to conduct highthroughput screens that interrogate phenotypes associatedwith the loss-of-function of many genes simultaneously. By suppressing gene expression and therefore protein function, RNAi, to a certain extent, models the pharmacological inhibition of a target protein and is therefore an effective tool for proof-of-principle experiments to identify and validate cancer drug targets.

[00109] The drug discovery process can be broadly summarized into five stages: target identification, target validation, high-throughput compound screening, lead optimization and clinical trials. The use of RNAi on cells cultured on user-defined substrate stiffnesss, and in particular high-throughput RNAi approaches, improves and enhances many of the stages of drug discovery, and in doing so streamline the process from the initial target identification to the development of a drug for use in the clinic. The substrate assay systems and methods here provide optimal fidelity to the clinical situation and provides a means of target identification to distinguish key targets that are essential for the survival and behavior of tumor cells in a clinically relevant setting but are redundant in normal cells by testing proliferation, cell morphology, cell mobility, invasion and metastasis, sustained angiogenesis, limitless replicative potential, the ability to evade apoptosis, insensitivity to anti-growth signals, and self sufficiency in growth signals.

Miscellaneous Agents

[00110] Some chemotherapy agents do not fall into the previous categories based on their activities. They include, but are not limited to, platinum coordination complexes, anthracenedione, substituted urea, methyl hydrazine derivative, adrenalcortical suppressant, amsacrine, L-asparaginase, and tretinoin. Platinum coordination complexes include such compounds as carboplatin and cisplatin (cis-DDP).

[00111] An anthracenedione such as mitoxantrone has been used for treating acute granulocytic leukemia and breast cancer. A substituted urea such as hydroxyurea has been used in treating chronic granulocytic leukemia, polycythemia vera, essential thrombocytosis and malignant melanoma. A methyl hydrazine derivative such as procarbazine (N- methylhydrazine, MIH) has been used in the treatment of Hodgkin's disease. An

adrenocortical suppressant such as mitotane has been used to treat adrenal cortex cancer, while aminoglutethimide has been used to treat Hodgkin's disease.

Definitions

[00112] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only not intended to be limiting. Other features and advantages of the invention will be apparent from the following detailed description and claims.

[00113] For the purposes of promoting an understanding of the embodiments described herein, reference will be made to preferred embodiments and specific language will be used to describe the same. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. As used throughout this disclosure, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a composition" includes a plurality of such compositions, as well as a single composition, and a reference to "a therapeutic agent" is a reference to one or more therapeutic and/or pharmaceutical agents and equivalents thereof known to those skilled in the art, and so forth.

[00114] Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[00115] The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or."

[00116] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. Examples

[00117] It is understood that modifications which do not substantially affect the activity of the various embodiments of this invention are also provided within the definition of the invention provided herein. Accordingly, the disclosed examples are intended to illustrate but not limit the present invention. While the claimed invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one of ordinary skill in the art that various changes and modifications can be made to the claimed invention without departing from the spirit and scope thereof. Thus, for example, those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention, and are covered by the following claims.

Example 1 : Molecular Screening Across Defined Stiffness Matrices Reveals Divergent Rho/RQCK Functions

[00118] The possibility that substrate stiffness fundamentally alters cellular responses to biochemical effectors has major implications for cell-based research, but remains largely unexplored. Therefore, we sought to integrate control of substrate stiffness into a standard multiwell format, emphasizing low cost, high throughput, and compatibility with

conventional biological assays and detection methods.

[00119] Starting with a stiffness-tunable polyacrylamide system (Pelham, R.J. & Wang, Y.L. Proc. Natl. Acad. Sci. U.S.A. 94, 1 3661-1 3665 (1997)), we developed a method to rapidly cast hydrogels of specified stiffness within multiwell, glass-bottom plates (Figure 4). The gels are optically transparent, thin (<100 μιη) and bound covalently to the entire surface of each well. Any configuration of wells encompassing the stiffness range of adipose tissue to skeletal muscle (shear moduli of 100-25,600 Pa) can be specified (Figs, la and lb). To enable cell attachment, the otherwise inert gels are functionalized with equivalent amounts monomelic collagen, ensuring that substrate modulus is the lone variable in the system (Figure la and Figure 5). With this method, over 100 plates can be assembled and made ready for cell culture within a single day. [00120] As an initial assessment of the platform's capabilities, we surveyed the effect of substrate stiffness on the accumulation (net change in cell number from 4 to 72 hours) of 10 cell types (Figure lc). Cells were seeded at a subconfluent density (15 cells/mm²) in 10% serum and cultured across five shear moduli spanning a broad, physiological range. After 72 hours, the majority of both normal and transformed cells exhibited generally increasing accumulation across stiffness, though some cell types were characterized by an abrupt increase in accumulation occurring at an intermediate stiffness. Strikingly, the growth of L292 and HEK29 3 cells was completely insensitive to stiffness, supporting the idea that the ability to detect or respond to mechanical signals may be lost in some cell types that have undergone malignant transformation (Wang, H.B., Dembo, M. & Wang, Y.L. Am. J.

Physiol., Cell Physiol. 279, CI 345-1 350 (2000)).

[00121] Selecting normal human lung fibroblasts for further study, we first considered how the density of gel-bound collagen might interact with stiffness to generate the accumulation profile. The effect of increasing collagen up to 12-fold over a minimally detectable threshold was relatively inconsequential, suggesting that stiffness overrides even substantial changes in ligand density (Figure Id). Next, we fixed collagen at an intermediate density (10 μg/ml applied collagen) and assessed the effect of varying serum concentration (Figure le). At 10% serum, cell accumulation was net positive across the entire stiffness range, and trending upward with increasing stiffness. The apparent promotion of cell proliferation was confirmed by the profile of BrdU incorporation across stiffness (Figure If). Reducing serum

concentration to 3% downshifted the growth curve to an extent that restricted positive cell accumulation to substrates >400 Pa. At 1 % serum, substrates <1600 Pa exhibited a net loss of cells, whereas on more rigid substrates cell number was maintained at the initial attachment density. On a log-log scale, the slope of the growth curves did not change appreciably under any of these circumstances, again consistent with a robust effect of stiffness on cell accumulation. The net loss of cells at low stiffness under reduced serum conditions was consistent with measurements of increasing apoptosis, as detected by caspase 3/7 activity, with decreasing matrix stiffness (Figure If). Together, these results emphasize the robust effect of increasing matrix stiffness on the promotion of cell proliferation and attenuation of apoptosis across a wide range of matrix density and serum contexts.

[00122] The ability to survey stiffness-specified biology at this level of precision enabled us to conduct a pharmacological screen of known or suspected inhibitors of cell proliferation. To increase throughput, we manufactured 96-well plates specifying only two contexts: the approximate shear modulus of lung parenchyma (400 Pa, F.L. et al.; unpublished data) and rigid glass. We seeded lung fibroblasts in 10% serum at a subconfluent density and exposed them to increasing doses of drugs for 72 hours. Overall, an unexpectedly diverse set of context-specific responses emerged from the evaluation of only 23 compounds (Figure 6a-c and Table 1). Not surprisingly, the effects of a number of compounds were indistinguishable between 400 Pa and glass (Figure 6a), exemplified by NSC 2 3766, an inhibitor of Racl (Figure 2a). However, the responses to a large subset of compounds subtly but significantly differed in magnitude between soft and rigid contexts (Figure 6b), as was the case for PD17 3074, an inhibitor of FGF receptor 1, and cantharidin, an inhibitor of protein phosphatases 1 and 2A (Figure 2a) Unexpectedly, treatment with blebbistatin significantly stimulated cell accumulation on 400 Pa, while simultaneously inhibiting accumulation on glass (Figure 2b).

[00123] Table 1-1 : Molecular actions of exemplary tested compounds and classes of compounds to be screened.

[00124] Because blebbistatin is a potent inhibitor of non-muscle myosin II, we further explored compounds that target known regulators of myosin II activity. Inhibition of Rho- associated kinase (ROCK) by GSK 429286 (Figure 2b), Y-27632, Fasudil hydrochloride and H-l 152 (Figure 7a), all succeeded in recapitulating the divergent, stiffness-specific response to blebbistatin. ROCK inhibition also rescued the cell spreading defect observed on 100 and 400 Pa substrates (Figure 8), consistent with its growth-promoting effects in these contexts. Similarly divergent responses were elicited when using C 3 transferase to block the GTP- bound form of RhoA upstream of ROCK, further confirming the matrix stiffness-dependent effect of this pathway on the regulation of cell proliferation (Figure 2b). While previous observations had identified a consensus obligate role for Rho and myosin-dependent tension in cell cycle progression (Wozniak, M.A. & Chen, C.S. Nat. Rev. Mol. Cell Biol. 10, 34-4 3 (2009)), these prior studies were largely confined to cells cultured on rigid substrates. The current findings, in contrast, indicate that the effects of Rho/ROCK on cell proliferation are surprisingly dependent on the matrix stiffness context.

[00125] To further explore the broader relevance of the stiffness-dependent effector function of this pathway, we surveyed the response of lung fibroblasts to Rho/ROCK/myosin II inhibition across five shear moduli (Figure 2c). With all drugs tested, growth stimulation was maximal at 400 Pa. Increasing matrix stiffness gradually dampened, and ultimately reversed, the growth enhancing effect. The incorporation of BrdU following treatment with 1 μΜ GSK 429286 largely mirrored this trend (Figure 2d). Conversely, GSK 429286 suppressed the activity of caspase 3/7 at 400 Pa, but progressively enhanced caspase 3/7 activity as matrix stiffness increased (Figure 2d). When we assessed the effect of ROCK inhibition on other cell types (Figs. 3a, 3b and Figure 7b), we found that the divergent growth response was fully recapitulated in human adipose-derived stem cells (hASC), human mesenchymal stem cells and NIH 3T 3 cells. However, the phenomenon appeared to be muted in MDCKII and A549 epithelial cells, as treatment with GSK 429286 elicited differences in the magnitude, but not the direction of the response. Notably, the response of HEK29 3 cells was indistinguishable on 400 Pa and glass, consistent with its stiffness- insensitive growth profile. To assess whether the divergent stiffness-dependent response to Rho/ROCK perturbations are characteristic of cells expressing a mesenchymal phenotype, we co-stimulated A549 cells with TGFI31, or induced epithelial-mesenchymal transition prior to seeding A549 cells in defined stiffness plates (Figure 3c). Both strategies succeeded in switching the effect of ROCK inhibition from growth attenuating to growth promoting on 400 Pa gels, whereas on glass, the character of the response was not fundamentally altered. Together these results demonstrate that divergent stiffness-dependent drug responses can be evoked across a physiologically relevant matrix stiffness range, and from a diverse set of human cell types expressing mesenchymal characteristics.

[00126] Methods

[00127] Multiwell plate fabrication. Glass-bottom, black-walled, 96-well plates (Matrical Biosciences) were treated with an aqueous solution of g-methacryloxypropyltrimethoxysilane (Acros Organics) for 1 hour, rinsed in distilled water and allowed to dry. Solutions containing 0.10% ammonium persulfate, 0.15% tetramethylethylenediamine and variable ratios of acrylamide: bisacrylamide (Bio-Rad) were delivered into selected wells of the multiwell plate. A custom built, 96-pin array with affixed borosilicate glass squares (Hausser Scientific) rendered hydrophobic by treatment with SurfaSil (Pierce) was used to sandwich the solutions between opposing glass surfaces and achieve a final gel thickness of < 100 μιη. After 10 minutes, the array was removed and the polymerized gels were rinsed with distilled water. To derivatize the gels, 50 μΐ of sulfo-SANPAH (G-Biosciences) at 50 μg/ml in 50 mM HEPES buffer, pH 8.5 was delivered to each well and activated by UV exposure. The solution was replaced with 100 μΐ of collagen (PureCol) in PBS at 10 μg/ml (unless otherwise indicated) and incubated at room temperature for 4 hours. The gels were rinsed with PBS and UV- sterilized prior to validation studies and cell seeding.

[00128] Hydrogel stiffness. Gels were mechanically characterized using an Asylum MFP- 3D atomic force microscope. Force-indentation profiles were acquired at an indentation rate of 20 μΓη/s using a sphere-tipped probe (Novascan) with a diameter of 5 μΜ and a nominal spring constant of -60 pN/nm. Young's modulus was calculated from fitting force-indentation data using a Hertz sphere model and converted to shear modulus using a Poisson's ratio of 0.3. [00129] Collagen density. Gels conjugated with various amounts of collagen were blocked with 1 % goat serum in PBS for 1 hour, and incubated for 2 hours with a mouse monoclonal antibody against native type I collagen (COL-1, Sigma) diluted 1 :250 in PBS. Gels were washed 3x with 0.1% Triton X-100 in PBS for 5 minutes each, and incubated for 1 hour with a goat anti-mouse HRP-conjugated antibody (Cell Signaling) diluted 1 : 1000 in PBS. The gels were washed 3x for 15 minutes each, and then 100 μΐ of Supersignal West Pico

Chemiluminescence Substrate (Pierce) was added to each well. Images were captured using a CCD camera (Syngene) within a linear range of detection, and average pixel densities were evaluated in Adobe Photoshop 6.0.

[00130] Cell culture and assays. For all experiments, normal human lung fibroblasts (Lonza) were used at passage 3-6. Human adipose-derived stem cells (ZenBio) and human mesenchymal stem cells (Tulane University) were used at passage 1- 3. L292 cells were purchased from ATCC, and all other cell types were generously provided by collaborators. All cells were cultured in Kaighn's Modification of Ham's F12 Medium (F12K)

supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin (all from Mediatech) in a humidified 37 °C incubator with 5% C02. For multiwell plate assays, the top and bottom rows served as cell-free background controls for each stiffness context represented. Relative cell numbers were assessed by the Cyquant NF Cell

Proliferation Assay (Invitrogen). To directly evaluate proliferation, relative amounts of incorporated bromodeoxyuridine (BrdU) were determined using a colorimetric, Cell Proliferation ELISA (Roche Applied Science) following a 24 hour exposure to BrdU.

Apoptosis was assessed using a fluorescence -based, ApoONE Caspase 3/7 Activity Assay (Promega).

[00131] Cell growth profiling. Cells were seeded in serum-free F12K media at -15 cells/mm² in multiwell plates specifying five stiffness contexts, and allowed to attach for 4 hours. Media was replaced with F12K containing 10% serum and cells were cultured for an additional 72 hours. For each stiffness condition, fold change was expressed as the ratio of adherent cell number at 72 versus 4 hours.

[00132] Epithelial-Mesenchymal Transition induction. Subconfluent A549 cells were cultured in media supplemented with 10 ng/ml of recombinant TGFj31 (R&D Systems) for 3 days, and withdrawn prior to cell seeding in multiwell plates. [00133] Drug screening. GF10920 3X, NSC2 3766, simvastatin, PP1 , FAK Inhibitor 14, SU9516, okadaic acid, cantharidin, taxol, IPA 3, cytochalasin D, calpeptin, Y-276 32, GSK429286 and fasudil hydrochloride were purchased from Tocris Bioscience;

dexamethasone and PD17 3074 from Sigma; H-1152, SP600125, cycloheximide, ML-7 and blebbistatin from Calbiochem; cell-membrane permeable C 3 transferase from Cytoskeleton, Inc. Cell numbers were evaluated 72 hours after a single addition of the indicated

concentrations of drugs.

[00134] Fluorescence cell staining. NHLFs in multiwell plates were fixed in 10% formalin and blocked in 1 % goat serum. F-actin and nuclei were stained with Alexa Fluor 488- Phalloidin and Hoescht 33342 (both from Invitrogen), respectively. Fluorescence images were captured with a Nikon TE 300 fluorescent microscope.

Example 2: Effect of knockdown of genes with RNAi

[00135] To mimic the effects of blebbistatin using a gene targeting approach, fibroblasts were treated with siRNA targeting non-muscle myosin heavy chain Ila (MYH9) or a scrambled siRNA control for 72 hours, then seeded on 400 Pa and rigid substrates and cultured for an additional 72 h. Figure 9 shows the fold change in cell number over the initial seeding density for each indicated concentration of siRNA. Results were normalized against the effect of negative control siRNA. MYH9 knockdown exerted divergent effects on growth promotion as matrix stiffness varied from rigid to 400 Pa gels, recapitulating the effect of blebbistatin.

Example 3: Automated fluorescent imaging in a 384 well plate

[00136] Automated fluorescent imaging was performed as follows. Cells in 384 well plates were imaged using a Pathway HT (AttoBioscience) fluorescence imaging system with autofocusingSignaling) diluted 1 : 1000 in PBS. The gels were washed 3x for 15

minutes each, and then 100 ul of Supersignal West Pico Chemiluminescence Substrate (Pierce) was added to each well. Images were captured using a CCD camera (Syngene) within a linear range of detection, and average pixel densities were evaluated in Adobe Photoshop 6.0.

[00137] To demonstrate compatibility with automated imaging systems, PA gels were fabricated spanning the entire stiffness range (0.3- 55 kPa) in a 384 well plate and seeded 7 cell types at various densities. After 24 hours in culture, the cells were fixed and stained to visualize f-actin and nuclei. Using autofocusing, we captured images of cells on all substrates, including glass (Figure 10). Prominent morphological transitions in the 1-6 kPa range were evident in many of the primary and immortalized cell lines, with the exception of L929 cells, which were virtually indistinguishable under all stiffness conditions.

[00138] The cells types shown in Figure 10 are provided in the table below.

OTHER EMBODIMENTS

[00139] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[00140] The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

[00141] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for screening for compounds that have cell proliferation modulating

activity comprising:

contacting a cell or cell population with a test agent, wherein the cell or cell population is adherent to a hydrogel having a shear modulus of about 10 to about 150,000 Pascals; and

determining whether cell proliferation activity is affected by the presence of the test agents as compared to a control.

2. A method for screening for compounds that have cell proliferation modulating activity comprising:

placing cells or cell populations in assigned locations in an array of compliant substrates having a shear modulus of between about 10 to about 150,000 Pascals;

contacting the cells or cell populations with a test agent; and

determining whether cell proliferation activity of any one of the cell or cell populations of the array is affected by the presence of the test agents as compared to a control.

3. A method for screening for compounds that inhibits cell proliferation comprising:

placing a cell or cell population in assigned locations in an array of compliant substrates having a shear modulus of about 10 to about 150,000 Pascals;

contacting the cell or cell population with a test agent; and

determining which cell or cell population populations are rendered sensitive to drug treatment, thereby identifying a drug that inhibits cell proliferation.

4. A method for screening for compounds that have cell adherence modulating activity comprising:

contacting a cell or cell population with a test agent, wherein the cell or cell population is adherent to a hydrogel having a shear modulus of about 10 to about 150,000 Pascals; and

determining whether cell adherence activity is affected by the presence of the test agents as compared to a control.

5. A method for screening for screening for compounds that have cell adherence modulating activity comprising: placing cells or cell populations in assigned locations in an array of compliant substrates having a shear modulus of between about 10 to about 150,000 Pascals;

contacting the cells or cell populations with a test agent; and

determining whether cell adherence activity of any one of the cell or cell populations of the array is affected by the presence of the test agents as compared to a control.

6. A method for screening for compounds that inhibits cell adherence comprising:

placing a cell or cell population in assigned locations in an array of compliant substrates having a shear modulus of about 10 to about 150,000 Pascals;

contacting the cell or cell population with a test agent; and

determining which cell or cell population populations are rendered sensitive to drug treatment, thereby identifying a drug that inhibits cell adherence.

7. A method for screening for compounds that have cell spreading modulating activity comprising:

contacting a cell or cell population with a test agent, wherein the cell or cell population is adherent to a hydrogel having a shear modulus of about 10 to about 150,000 Pascals; and

determining whether cell spreading activity is affected by the presence of the test agents as compared to a control.

8. A method for screening for screening for compounds that have cell spreading modulating activity comprising:

placing cells or cell populations in assigned locations in an array of compliant substrates having a shear modulus of between about 10 to about 150,000 Pascals;

contacting the cells or cell populations with a test agent; and

determining whether cell spreading activity of any one of the cell or cell populations of the array is affected by the presence of the test agents as compared to a control.

9. A method for screening for compounds that inhibits cell spreading comprising:

placing a cell or cell population in assigned locations in an array of compliant substrates having a shear modulus of about 10 to about 150,000 Pascals;

contacting the cell or cell population with a test agent; and

determining which cell or cell population populations are rendered sensitive to drug treatment, thereby identifying a drug that inhibits cell spreading.

10. A method for screening for compounds that have apoptotic modulating activity comprising:

contacting a cell or cell population with a test agent, wherein the cell or cell population is adherent to a hydrogel having a shear modulus of about 10 to about 150,000 Pascals; and

determining whether cell viability is affected by the presence of the test agents as compared to a control.

11. A method for screening for screening for compounds that have apoptotic modulating activity comprising:

placing cells or cell populations in assigned locations in an array of compliant substrates having a shear modulus of between about 10 to about 150,000 Pascals;

contacting the cells or cell populations with a test agent; and

determining whether cell viability of any one of the cell or cell populations of the array is affected by the presence of the test agents as compared to a control.

12. A method for screening for compounds that induces apoptosis comprising:

placing a cell or cell population in assigned locations in an array of compliant substrates having a shear modulus of about 10 to about 150,000 Pascals;

contacting the cell or cell population with a test agent; and

determining which cell or cell population populations are rendered sensitive to drug treatment, thereby identifying a drug that induces apoptosis.

13. The method of any one of claims 10-12, wherein the test agent is known to induce

apoptosis in cells.

14. A method of performing automated fluorescent imaging comprising

placing a cell or cell population in assigned locations in an array of compliant substrates having a shear modulus of about 10 to about 150,000 Pascals;

fixing said cell or cell population;

staining said cell or cell population;

imaging said cell or cell population; and

detecting and processing cell images,

thereby performing automated fluorescent imaging.

15. The method of claim 14, wherein said cell or cell population is imaged using

autofocusing.

16. The method of claim 14, wherein each location in said array of compliant substrates comprises a unique shear modulus.

17. The method of claim 14, wherein imaging said cell or cell population comprises imaging cellular morphology.

18. The method of claim 14, wherein said array comprises a 384 well plate.

19. A method for screening for compounds that modulate cellular phenotype comprising: placing cells or cell populations in assigned locations in an array of compliant substrates having a shear modulus of between about 10 to about 150,000 Pascals;

contacting said cells or cell populations with a nucleic acid composition; and determining whether cellular phenotype of any one of the cell or cell populations of the array is affected by the presence of the nucleic acid composition as compared to a control.

20. The method of claim 19, wherein said phenotype comprises cellular morphology, cellular adhesion, or cytoskeleton function.

21. The method of claim 19, wherein said nucleic acid composition is selected from the group consisting of a small interfering RNA (siRNA) and a short hairpin RNA (shRNA).

22. A method of determining an individualized course of treatment of a subject comprising: providing a cell population from said subject;

placing said cells or cell population in assigned locations in an array of compliant substrates having a shear modulus of between about 10 to about 150,000 Pascals;

contacting said cells or cell population with a test agent;

determining whether cell viability, cell proliferation activity, adherence activity, or spreading activity of any one of the cells or cell populations of the array is increased or reduced by the presence of the test agent compared to a control.

23. The method of claim 22, wherein said individualized course of treatment is for cancer, hypertension, or fibrosis.